花青素相关论文.docx

1、花青素相关论文Food and Chemical Toxicology 43 (2005) 15571566E ects of anthocyanidin on the inhibition of proliferation and induction of apoptosis in human gastric adenocarcinoma cellsPing-Hsiao Shih, Chi-Tai Yeh, Gow-Chin Yen *Department of Food Science, National Chung Hsing University, 250 Kuokuang Road,

2、 Taichung 40227, TaiwanReceived 15 December 2004; accepted 4 May 2005AbstractAnthocyanins are naturally occurring reddish pigments that abundant in fruits and vegetables. To investigate the mechanistic basis for the anti-tumor properties of anthocyanins, five aglycone (cyanidin, delphinidin, malvidi

3、n, pelargonidin, and peonidin) and four glycosylated (cyaniding-3-glucoside, malvidin-3-glucoside, pelargonidin-3-glucoside and peonidin-3-glucoside) anthocyanins were used to examine their e ects on cell cycle progression and induction of apoptosis in human gastric adenocarcinoma AGS cells. The dat

4、a from cell viability assay showed that malvidin exhibited the most potent anti-proliferation e ect on AGS cells in a time-and dose-dependent manner (P 0.05). This event is accompanied the arrest of AGS cells at the G0/G1 phase by malvidin at the tested concentrations of 0200 lM. Cellular uptake of

5、anthocyanin and anthocyanidin was confirmed by HPLC analysis and the intracellular accumulation of malvidin (24.9 1.1 lM/mg protein) was observed when treatment of AGS cells with malvidin for 12 h. In addition, an accumulation of AGS cells in sub-G1 phase (20% and 30% increase for 100 and 200 lM of

6、malvidin, respectively) was observed as well as by the appearance of a fraction of cells with an aneudiploid DNA content. The occurrence of apoptosis induced by malvidin was confirmed by morphological and biochemical features, including apoptotic bodies formation, caspase-3 acti-vation and poly(ADP-

7、ribose) polymerase proteolysis. Furthermore, the mitochondrial membrane potential of apoptotic cells after treatment with malvidin was significantly lost and resulted in the elevation of Bax/Bcl-2 ratio for 1.6-fold against control for100lM treatment. In addition, the malvidin treatment significantl

8、y increased the p38 kinase expression and inhibited the ERK activ-ity, and the e ects of malvidin on caspase-3 activation were blocked, respectively, by the ERK and p38 inhibitors. These findingssuggest that growth inhibition and cytotoxicity of AGS cells by malvidin is involved in the induction of

9、apoptosis rather than necrosis.2005 Elsevier Ltd. All rights reserved.Keywords: Anthocyanidin; Apoptosis; AGS cells; MAPK pathway; Bcl-2 family1. IntroductionFollowing the change of dietary habits and the exces-sive process of food, the tendency of gastrointestinal dis-ease is on the rise. Gastric c

10、ancer is a kind of gastrointestinal (GI) tract cancer that is the leading cause of cancer-related mortality in the world and approximately 90% of stomach cancers are adenocarci-*Corresponding author. Tel.: +886 4 2287 9755; fax: +886 4 2285 4378.E-mail address: gcyendragon.nchu.edu.tw (G.-C. Yen).02

11、78-6915/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2005.05.001nomas (Kelley and Duggan, 2003). Studies have shown that a high intake of smoked, salted, nitrated foods and carbohydrates, but a low intake of vegetables, fruits, and milks, are linked to cancer incide

12、nce. These diets have been shown to significantly increase the risk for stomach cancer (Serafini et al., 2002). Epidemiological studies have provided convincing evidence that dietary factors can modify the process of carcinogenesis, includ-ing initiation, promotion and progression of several types o

13、f human cancer (Ray, 2005). The occurrence of gastrointestinal (GI) cancers has increased strikingly during the past decade. Despite advances in early1558 P.-H. Shih et al. / Food and Chemical Toxicology 43 (2005) 15571566diagnosis and treatment modalities, the side e ects of chemical drugs and recu

14、rrence are still the problems. Therefore, the development of chemotherapeutic/che-mopreventive agents for gastric cancer is important for reducing the mortality caused by this disease.Anthocyanins are glycosides of anthocyanidins univer-sally associated with attractive, colorful, and flavorful fruit

15、s. Recently, there has been a resurgence of interest in anthocyanins due to their potential biological and phar-macology benefit, such as: antioxidant (Moyer et al., 2002), anti-inflammatory (Subarnas and Wagner, 2000), reducing the risk of cardiovascular diseases (Wang and Mazza, 2002), and anti-tu

16、mor properties (Katsube et al., 2003). Animal studies have also demonstrated that feeding with anthocyanin-rich extract protected against tert-butyl hydroperoxide-induced hepatic toxicity (Wang et al., 2000) and decrease lipid peroxidation and DNA damage in vitamin E-depleted rats (Ramirez-Tortosa e

17、t al., 2001). More recently, anthocyanins have been shown to be an e ective chemopreventive agent against 1,2-dimethyhydrazine- and 2-amino-1-methyl-6-phenylimi-dazo4,5-bpyridine-induced mammary carcinogenesis in rats (Hagiwara et al., 2002). In addition, anthocyanins can be directly absorbed and di

18、stributed to the blood (Tsuda et al., 1999), and to be incorporated in cell cultures, both in the plasma membrane and in the cytosol (Youdim et al., 2000). Extensive studies indicate that anthocyanins have strong free radical scavenging and antioxidant activi-ties (Wang et al., 1997), suggesting tha

19、t they play an important role in preventing against mutagenesis and car-cinogenesis (Omenn, 1995). Anthocyanins also showed inhibitory e ects on the growth of some cancer cells (Kamei et al., 1995). Our recent study has reported (Yeh and Yen, 2005) that the induction of apoptosis by the anthocyanidi

20、ns through regulation of Bcl-2 gene and activation of c-jun n-terminal kinase cascade in hepatoma cells. However, the research concerning the protective e ect of anthocyanins against gastric adenocarcinoma is limited; thus, the objective of the present study was to determine whether anthocyanins wou

21、ld be useful in the prevention of human gastric adenocarcinoma car-cinogenesis. In the present study, human cultured gastric adenocarcinoma (AGS) cells were chosen for study.AGS cells line has been shown to grow in athymic mice and to have the same histochemical and cytological characteristics as th

22、e specimen taken from the patient. It is important to characterize human tumor cells in vitro in this detailed manner, since they serve as excellent model systems for other studies involving the heterogeneous re-sponses to anticancer drugs (Barranco et al., 1983), and recently this cell line has bee

23、n widely used as a model sys-tem for evaluating cancer cell apoptosis (Liu et al., 2003).Apoptosis is a defined type of cell death and di ers from traditional cell death, necrosis. Many recent stud-ies have indicated that anticancer drugs or cancer che-mopreventive agents act through the induction o

24、fapoptosis to prevent tumor promotion, progression, and the occurrence of cellular inflammatory responses other than necrosis. Apoptosis is also a gene-directed form of cell death with well-characterized morpholo-gical and biochemical features (Brown and Attardi, 2005). Initiation of apoptosis appea

25、rs to be a common mechanism of many cytotoxic agents used in chemother-apy. Therefore, apoptosis inducing agents are expected to be ideal anticancer drugs. In addition, some flavo-noids, such as quercetin, apigenin, and phloretin, inhibit cancer cell growth through the induction of apoptosis (Wang e

26、t al., 1999). Therefore, understanding the mech-anism of apoptosis has important implications in the prevention and treatment of many diseases.In this study, we investigated the induction of apop-tosis by five typical anthocyanins and their relative antho-cyanidins in human gastric adenocarcinoma ce

27、lls. In addition, the molecular mechanisms of the apoptotic e ects induced by malvidin were also determined.2. Materials and methods2.1. MaterialsAnthocyanidins cyanidin chloride, delphinidin chloride, malvidin chloride, pelargonidin chloride, peonidin chloride, anthocyanins cyanidin-3-O-gluco-side

28、chloride, malvidin-3-O-glucoside chloride (oenin), pelargonidin-3-O-glucoside chloride, and peonidine-3-O-glucoside chloride were obtained from Extrasynthese (Genay, France). Human adenocarcinoma AGS cells were purchased from the Bioresource Collection and Research Center (BCRC, Food Industry Resear

29、ch and Development Institute, Hsin Chu, Taiwan). Goat anti-rabbit IgG polyclonal antibody conjugated to peroxidase were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Anti-ERK, anti-phospho-ERK, anti-JNK, anti-phospho-JNK, anti-p38, anti-phospho-p38, anti-capsase-3, anti-poly (ADP-ribose) po

30、lymerase, and anti-b-actin antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-Bcl-2, and anti-Bax antibodies were obtained from Pharmingen (San Diego, CA). Molecular mass markers for proteins were obtained from Pharmacia Biotech (Saclay, France). Polyvinyldi-fluoride (PVDF)

31、membrane for Western blotting was obtained from Millipore (Bedford, MA, USA).2.2. Cell cultureThe AGS cells were cultured in Ham s F-12K medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin,100lg/ml streptomycin) at 37 LC in a humidified atmo-sp

32、here of 5% CO2. During the experiments the concen-tration of FBS were adjusted to 5%.P.-H. Shih et al. / Food and Chemical Toxicology 43 (2005) 1557156615592.3. Cell morphological observationMorphology of control cells and samples-treated cells were detected by phase contrast microscope (Nicon).2.4. Cell viability assayCell viability assay was performed with MTT photo-metric analysis and trypan blue dye exclusion method. In the MTT assay, 5 104 cells were seeded in 96-well microtiter plate. Cells were treated without or with va-rious concen